Altered Conformational State Research Articles

Structural insights into SARS-CoV-2 main protease conformational plasticity.

The spread of different severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) variants underscores the need for insights into the structural properties of its structural and non-structural proteins. The highly conserved homo-dimeric chymotrypsin-like protease (3CL MPRO ), belonging to the class of cysteine hydrolases, plays an indispensable role in processing viral polyproteins that are involved in viral replication and transcription. Studies have successfully demonstrated the role of MPRO as an attractive drug target for designing antiviral treatments because of its importance in the viral life cycle. Herein, we report the structural dynamics of six experimentally solved structures of MPRO (i.e.,6LU7, 6M03, 6WQF, 6Y2E, 6Y84, and 7BUY including both ligand-free and ligand-bound states) at different resolutions. We have employed a structure-based balanced forcefield, CHARMM36m through state-of-the-art all-atoms molecular dynamics simulations at µ-seconds scale at room temperature(303K) and pH 7.0 to explore their structure-function relationship. The helical domain-III responsible for dimerization mostly contributes to the altered conformational states and destabilization of MPRO . A keen observation of the high degree of flexibility in the P5 binding pocket adjoining domain II-III highlights the reason for observation of conformational heterogeneity among the structural ensembles of MPRO . We also observe a differential dynamics of the catalytic pocket residues His41, Cys145, and Asp187, which may lead to catalytic impairment of the monomeric proteases. Among the highly populated conformational states of the six systems, 6LU7 and 7M03 forms the most stable and compact MPRO conformation with intact catalytic site and structural integrity. Altogether, our findings from this extensive study provides a benchmark to identify physiologically relevant structures of such promising drug targets for structure-based drug design and discovery of potent drug-like compounds having clinical potential.

Allosteric inhibition of PPM1D serine/threonine phosphatase via an altered conformational state

PPM1D encodes a serine/threonine phosphatase that regulates numerous pathways including the DNA damage response and p53. Activating mutations and amplification of PPM1D are found across numerous cancer types. GSK2830371 is a potent and selective allosteric inhibitor of PPM1D, but its mechanism of binding and inhibition of catalytic activity are unknown. Here we use computational, biochemical and functional genetic studies to elucidate the molecular basis of GSK2830371 activity. These data confirm that GSK2830371 binds an allosteric site of PPM1D with high affinity. By further incorporating data from hydrogen deuterium exchange mass spectrometry and sedimentation velocity analytical ultracentrifugation, we demonstrate that PPM1D exists in an equilibrium between two conformations that are defined by the movement of the flap domain, which is required for substrate recognition. A hinge region was identified that is critical for switching between the two conformations and was directly implicated in the high-affinity binding of GSK2830371 to PPM1D. We propose that the two conformations represent active and inactive forms of the protein reflected by the position of the flap, and that binding of GSK2830371 shifts the equilibrium to the inactive form. Finally, we found that C-terminal truncating mutations proximal to residue 400 result in destabilization of the protein via loss of a stabilizing N- and C-terminal interaction, consistent with the observation from human genetic data that nearly all PPM1D mutations in cancer are truncating and occur distal to residue 400. Taken together, our findings elucidate the mechanism by which binding of a small molecule to an allosteric site of PPM1D inhibits its activity and provides insights into the biology of PPM1D.

Structural insights of metallo-beta-lactamase revealed an effective way of inhibition of enzyme by natural inhibitors

Metallo-beta-lactamase (MBL) is a class of enzyme that catalyzes the hydrolysis of a broad range of beta-lactam antibiotics leading to the development of drug resistance in bacteria. Inhibition of MBL is therefore pursued as a potential way to increase the susceptibility of bacteria to beta-lactam antibiotics. In this study, MBL inhibitors from natural sources such as Eupalitin, Rosmarinic acid and Luteolin are used as a potential alternative to explore their effect. The crystal structure of MBL revealed a hydrolyzed Meropenem, which was undocked from the active center pocket to get the apo-protein. The apo-protein was re-docked with substrate, three known MBL inhibitors and natural compounds to prepare the starting structure in the current work and to draw conclusions. Further, to explore the efficiency of natural inhibitors, we analyzed the dynamic behavior of the enzyme over simulation time using molecular dynamics studies. Our results suggest that MBL enzyme adopted altered conformational state in the presence of natural inhibitor. This is because, the natural inhibitors were tried to occupy a different binding pocket in the enzyme by causing positional drift from the active center pocket. Here, the different binding pocket partly comprised of active site pocket and partly by a new region explored by ligand, making it inappropriate for substrate to occupy the active site. Thus natural inhibitors may be potential entities to target MBL.AbbreviationsADMEAbsorption, Distribution, Metabolism and ExcretionBBBBlood brain barrierCHARMMChemistry at Harvard Macromolecular MechanicsCOMCenter of MassCYP2D6Cytochrome P450 2D6DSDiscovery StudioESBLExtended Spectrum Beta-lactamasesFDAFood and Drug AdministrationGLASSGlobal antimicrobial resistance surveillance systemGROMACSGROningen MAchine for Chemical SimulationsKDEKernel Density Estimation PlotsMBLMetallo-beta-lactamaseMBL-CMetallo-beta-lactamase bound to L-CaptoprilMBL-EMetallo-beta -lactamase bound to EupalitinMBL-IMetallo-beta -lactamase bound to ImipenemMBL-LMetallo-beta -lactamase bound to LuteolinMBL-RMetallo-beta -lactamase bound to Rosmarinic acidMDMolecular DynamicsMMPBSAMolecular Mechanics Poisson − Boltzmann surface areaNPTNumber of atoms in the system, Pressure of the system and Temperature of the systemnsNano secondsNVTNumber of atoms in the system, Volume of the system, and Temperature of the systemPDBProtein Data BankRgRadius of GyrationRMSDRoot Mean Square DeviationRMSFRoot Mean Square FluctuationSASASolvent Accessible Surface AreaSPC/ESimple Point ChargeWHOWorld Health OrganizationCommunicated by Ramaswamy H. Sarma

Star‐Shaped Gold Nanoparticles as Friendly Interfaces for Protein Electrochemistry: the Case Study of Cytochrome c

AbstractGold nanostars with an average tip‐to‐tip length of 52±6 nm were functionalized with different capping agents and used as electrode modification materials for protein electrochemistry. Direct electron transfer between cytochrome c and nanostar‐coated pyrolytic graphite electrodes was observed with the protein in solution. The electrochemical response was improved at nanostars functionalized with a 1 : 1 mixture of 11‐mercaptoundecanoic acid and 4‐mercaptobenzoic acid in comparison with gold nanospheres coated with a similar functionalization. Further immobilization of cytochrome c on pyrolytic graphite while conjugated with the same nanostars guaranteed the maintenance of the protein's native properties, whereas direct adsorption on the bare or nanostar‐modified electrodes resulted in an altered conformational state. The pseudo‐peroxidase activity of the altered cytochrome c was enhanced in the presence of the nanostars.

Transporters through the looking glass. An insight into the mechanisms of ion-coupled transport and methods that help reveal them.

Cell membranes, despite providing a barrier to protect intracellular constituents, require selective gating for influx of important metabolites including ions, sugars, amino acids, neurotransmitters and efflux of toxins and metabolic end-products. The machinery involved in carrying out this gating process comprises of integral membrane proteins that use ionic electrochemical gradients or ATP hydrolysis, to drive concentrative uptake or efflux. The mechanism through which ion-coupled transporters function is referred to as alternating-access. In the recent past, discrete modes of alternating-access have been described with the elucidation of new transporter structures and their snapshots in altered conformational states. Despite X-ray structures being the primary sources of mechanistic information, other biophysical methods provide information related to the structural dynamics of these transporters. Methods including EPR and smFRET, have extensively helped validate or clarify ion-coupled transport mechanisms, in a near-native environment. This review seeks to highlight the mechanistic details of ion-coupled transport and delve into the biophysical tools and methods that help in understanding these fascinating molecules.

Zyxin is a novel target for beta‐amyloid peptide: characterization of its role in Alzheimer's pathogenesis

Zyxin is an adaptor protein recently identified as a novel regulator of the homeodomain-interacting protein kinase 2 (HIPK2)-p53 signaling in response to DNA damage. We recently reported an altered conformational state of p53 in tissues from patients with Alzheimer 's disease (AD), because of a deregulation of HIPK2 activity, leading to an impaired and dysfunctional response to stressors. Here, we examined the molecular mechanisms underlying the deregulation of HIPK2 activity in two cellular models, HEK-293 cells and SH-SY5Y neuroblastoma cells differentiated with retinoic acid over-expressing the amyloid precursor protein, focusing on the evidence that zyxin expression is important to maintain HIPK2 protein stability. We demonstrated that both beta-amyloid (Aβ) 1-40 and 1-42 induce zyxin deregulation, thus affecting the transcriptional repressor activity of HIPK2 onto its target promoter, metallothionein 2A, which is in turn responsible for the induction of an altered conformational state of p53. We demonstrate for the first time that zyxin is a novel target of Aβ activities in AD. These results may help the studies on the pathogenesis of AD, through the fine dissection of events related to beta-amyloid activities.

Derepression of Bacterial Transcription-Repair Coupling Factor Is Associated with a Profound Conformational Change

Transcription-repair coupling factor (TRCF; the product of the mfd gene) is a widely conserved bacterial protein that couples DNA repair with transcription. TRCF recognizes RNA polymerase stalled at a noncoding lesion in the DNA template strand, uses the energy from ATP hydrolysis to disrupt the transcription complex, and stimulates DNA repair by recruiting UvrA, a component of the nucleotide excision repair machinery, to the site. TRCF is a large (130 kDa) multifunctional protein with a complex structure–function relationship consisting of a compact arrangement of eight structured domains linked by flexible linkers. Through a conserved, intramolecular, interdomain interaction, TRCF is held in a conformation in which its enzymatic activities (ATPase activity and DNA translocase activity) are strongly repressed. Disruption of the repressive interdomain interaction by amino acid substitutions within the interface derepresses ATPase and DNA translocase activities. In this work, we have shown that derepressed TRCF mutants are dramatically sensitized to limited proteolysis compared with repressed TRCF, pointing to an altered conformational state. Analysis of the protease cleavage sites mapped onto the structure of the repressed TRCF conformation indicates that (1) the cleavage sites tend to cluster at linkers connecting the TRCF structured domains, and (2) many of the cleavage sites sensitized in the derepressed TRCF are partially or completely buried to protease access in the repressed TRCF structure. We conclude that TRCF derepression is associated with profound conformational changes that primarily involve a reorganization of the interdomain interactions.

Unfolded p53 in the pathogenesis of Alzheimer's disease: is HIPK2 the link?

p53 transcriptional activity depends mainly on posttranslational modifications and protein/protein interaction. Another important mechanism that controls p53 function is its conformational stability since p53 is an intrinsically unstable protein. An altered conformational state of p53, independent from point mutations, has been reported in tissues from patients with Alzheimer's disease (AD), leading to an impaired and dysfunctional response to stressors. Recent evidence shows that one of the activators that induces p53 posttranslational modification and wild-type conformational stability is homeodomain interacting protein kinase 2 (HIPK2). Hence, conditions that induce HIPK2 deregulation would result in a dysfunctional response to stressors by affecting p53 activity. Discovering the mechanisms of HIPK2 activation/inhibition and the ways to manipulate HIPK2 activity are an interesting option to affect several biological pathways, including those underlying AD. Soluble beta-amyloid peptides have recently been involved in HIPK2 degradation, in turn regulating the p53 conformational state and vulnerability to a noxious stimulus, before triggering the amyloidogenic cascade. Here we discuss about these findings and the potential relevance of HIPK2 as a target for AD and highlight the existence of a novel amyloid-based mechanism in AD potentially leading to the survival of injured dysfunctional cells.

Morpheeins – A New Pathway For Allosteric Drug Discovery~!2010-02-12~!2010-05-21~!2010-06-08~!

The morpheein model of allosteric regulation can be applied as a novel approach to the discovery of small molecule allosteric modulators of protein function. Morpheeins are homo-oligomeric proteins where, under physiological conditions, the oligomer can dissociate, the dissociated units can change conformation, and the altered conformational state can reassociate to a structurally and functionally distinct oligomer. This phenomenon serves as a basis for allostery, as a basis for conformational diseases, as a basis for drug discovery, and may be applicable to personalized medicine such as in the prediction of drug side effects. Each of these relationships has been established for the prototype morpheein, porphobilinogen synthase, where the conformational disease is a porphyria and the drug application is in antimicrobial discovery. These data are presented along with a discussion of other drug targets for which the morpheein model of allostery may apply. Such targets include HIV integrase, TNFα, β-tryptase, and p53.

Homeodomain Interacting Protein Kinase 2: A Target for Alzheimer's Beta Amyloid Leading to Misfolded p53 and Inappropriate Cell Survival

BackgroundHomeodomain interacting protein kinase 2 (HIPK2) is an evolutionary conserved serine/threonine kinase whose activity is fundamental in maintaining wild-type p53 function, thereby controlling the destiny of cells when exposed to DNA damaging agents. We recently reported an altered conformational state of p53 in tissues from patients with Alzheimer's Disease (AD) that led to an impaired and dysfunctional response to stressors.Methodology/Principal FindingsHere we examined the molecular mechanisms underlying the impairment of p53 activity in two cellular models, HEK-293 cells overexpressing the amyloid precursor protein and fibroblasts from AD patients, starting from recent findings showing that p53 conformation may be regulated by HIPK2. We demonstrated that beta-amyloid 1–40 induces HIPK2 degradation and alters HIPK2 binding activity to DNA, in turn regulating the p53 conformational state and vulnerability to a noxious stimulus. Expression of HIPK2 was analysed by western blot experiments, whereas HIPK2 DNA binding was examined by chromatin immunoprecipitation analysis. In particular, we evaluated the recruitment of HIPK2 onto some target promoters, including hypoxia inducible factor-1α and metallothionein 2A.Conclusions/SignificanceThese results support the existence of a novel amyloid-based pathogenetic mechanism in AD potentially leading to the survival of injured dysfunctional cells.

Cocaine produces D 2R-mediated conformational changes in the adenosine A 2AR-dopamine D 2R heteromer

Adenosine A 2A receptors (A 2ARs) and dopamine D 2 receptors (D 2Rs) form constitutive heteromers in living cells and exhibit a strong functional antagonistic interaction. Recent findings give neurochemical evidence that extended cocaine self-administration in the rat give rise to an up-regulation of functional A 2ARs in the nucleus accumbens that return to baseline expression levels during cocaine withdrawal. In the present work, the acute in vitro effects of a concentration of cocaine known to fully block the dopamine (DA) transporter without exerting any toxic actions were investigated on A 2AR and D 2LR formed heteromers in transiently co-transfected HEK-293T cells. In vitro treatment of cocaine was found to produce changes in D 2R homodimers and in A 2AR–D 2R heterodimers detected through bioluminescent energy transfer (BRET). Cocaine was found to produce a time- and concentration-dependent reduction in the BRET max between A 2AR–D 2LR heterodimers and D 2LR homodimers, but not A 2AR homodimers, indicating its effect on D 2R. Cocaine was evaluated with regard to D 2R binding using a human D 2LR stable expressing CHO cell line and was found to produce an increase in the affinity of hD 2LR for DA. At the level of G protein-coupling, cocaine produced a small, but significant increase in DA-stimulated binding of GTPγS. However, cocaine failed to modulate D 2R agonist-induced inhibition of cAMP in stable hD 2LR CHO cells or the gating of GIRK channels in oocytes. Taken together, these results indicate a direct and specific effect of a moderate concentration of cocaine on the DA D 2LR, that results in enhanced agonist recognition, G protein-coupling and an altered conformational state of D 2R homodimers and A 2AR–D 2R heterodimers.

B Cell Signaling and Fate Decision

Antigen receptors on the surface of B lymphocytes trigger adaptive immune responses after encountering their cognate antigens but also control a series of antigen-independent checkpoints during B cell development. These physiological processes are regulated by the expression and function of cell surface receptors, intracellular signaling molecules, and transcription factors. The function of these proteins can be altered by a dynamic array of post-translational modifications, using two interconnected mechanisms. These modifications can directly induce an altered conformational state in the protein target of the modification itself. In addition, they can create new binding sites for other protein partners, thereby contributing to where and when such multiple protein assemblies are activated within cells. As a new type of post-transcriptional regulator, microRNAs have emerged to influence the development and function of B cells by affecting the expression of target mRNAs.

Human wild-type alanine:glyoxylate aminotransferase and its naturally occurring G82E variant: functional properties and physiological implications

Human hepatic peroxisomal AGT (alanine:glyoxylate aminotransferase) is a PLP (pyridoxal 5'-phosphate)-dependent enzyme whose deficiency causes primary hyperoxaluria Type I, a rare autosomal recessive disorder. To acquire experimental evidence for the physiological function of AGT, the K(eq),(overall) of the reaction, the steady-state kinetic parameters of the forward and reverse reactions, and the pre-steady-state kinetics of the half-reactions of the PLP form of AGT with L-alanine or glycine and the PMP (pyridoxamine 5'-phosphate) form with pyruvate or glyoxylate have been measured. The results indicate that the enzyme is highly specific for catalysing glyoxylate to glycine processing, thereby playing a key role in glyoxylate detoxification. Analysis of the reaction course also reveals that PMP remains bound to the enzyme during the catalytic cycle and that the AGT-PMP complex displays a reactivity towards oxo acids higher than that of apoAGT in the presence of PMP. These findings are tentatively related to possible subtle rearrangements at the active site also indicated by the putative binding mode of catalytic intermediates. Additionally, the catalytic and spectroscopic features of the naturally occurring G82E variant have been analysed. Although, like the wild-type, the G82E variant is able to bind 2 mol PLP/dimer, it exhibits a significant reduced affinity for PLP and even more for PMP compared with wild-type, and an altered conformational state of the bound PLP. The striking molecular defect of the mutant, consisting in the dramatic decrease of the overall catalytic activity (approximately 0.1% of that of normal AGT), appears to be related to the inability to undergo an efficient transaldimination of the PLP form of the enzyme with amino acids as well as an efficient conversion of AGT-PMP into AGT-PLP. Overall, careful biochemical analyses have allowed elucidation of the mechanism of action of AGT and the way in which the disease causing G82E mutation affects it.

Activation of brain calcineurin (Cn) by Cu-Zn superoxide dismutase (SOD1) depends on direct SOD1–Cn protein interactions occurringin vitroandin vivo

Cn (calcineurin) activity is stabilized by SOD1 (Cu-Zn superoxide dismutase), a phenomenon attributed to protection from superoxide (O2*-). The effects of O2*- on Cn are still controversial. We found that O2*-, generated either in vitro or in vivo did not affect Cn activity. Yet native bovine, recombinant human or rat, and two chimaeras of human SOD1-rat SOD1, all activated Cn, but SOD2 (Mn-superoxide dismutase) did not affect Cn activity. There was also a poor correlation between SOD1 dismutase activity and Cn activation. A chimaera of human N-terminal SOD1 and rat C-terminal SOD1 had little detectable dismutase activity, yet stimulated Cn activity the same as full-length human or rat SOD1. Nevertheless, there was evidence that the active site of SOD1 was involved in Cn activation based on the loss of activation following chelation of Cu from the active site of SOD1. Also, SOD1 engaged in the catalysis of O2*- dismutation was ineffective in activating Cn. SOD1 activation of Cn resulted from a 90-fold decrease in phosphatase K(m) without a change in V(max). A possible mechanism for the activation of Cn was identified in our studies as the prevention of Fe and Zn losses from the active site of Cn, suggesting a conformation-dependent SOD1-Cn interaction. In neurons, SOD1 and Cn were co-localized in cytoplasm and membranes, and SOD1 co-immunoprecipitated with Cn from homogenates of brain hippocampus and was present in immunoprecipitates as large multimers. Pre-incubation of pure SOD1 with Cn caused SOD1 multimer formation, an indication of an altered conformational state in SOD1 upon interaction with Cn.

Probing the Structure of the Infectious Amyloid Form of the Prion-forming Domain of HET-s Using High Resolution Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

The HET-s prion protein of Podospora anserina represents a valuable model system to study the structural basis of prion propagation. In this system, prion infectivity can be generated in vitro from a recombinant protein. We have previously identified the region of the HET-s protein involved in amyloid formation and prion propagation. Herein, we show that a recombinant peptide corresponding to the C-terminal prion-forming domain of HET-s (residues 218-289) displays infectivity. We used high resolution hydrogen/deuterium exchange analyzed by mass spectrometry to gain insight into the structural organization of this infectious amyloid form of the HET-s-(218-289) protein. Deuterium incorporation was analyzed by ion trap mass spectrometry for 76 peptides generated by pepsin proteolysis of HET-s-(218-289). By taking into account sequence overlaps in these peptides, a resolution ranging from 4-amino acids stretches to a single residue could be achieved. This approach allowed us to define highly protected regions alternating with more accessible segments along the HET-s-(218-289) sequence. The HET-s-(218-289) fibrils are thus likely to be organized as a succession of beta-sheet segments interrupted by short turns or short loops.

Evidence that Vector Transmission of a Plant Virus Requires Conformational Change in Virus Particles

Transmission of Cucumber necrosis virus (CNV) by zoospores of its fungal vector, Olpidium bornovanus, involves specific adsorption of virus particles onto the zoospore plasmalemma prior to infestation of cucumber roots by virus-bound zoospores. Previous work has shown that specific components of both CNV and zoospores are required for successful CNV/zoospore recognition. Here, we show that limited trypsin digestion of CNV following in vitro CNV/zoospore binding assays, results in the production of specific proteolytic digestion products under conditions where native CNV is resistant. The proteolytic digestion pattern of zoospore-bound CNV was found to be similar to that of swollen CNV particles produced in vitro, suggesting that zoospore-bound CNV is in an altered conformational state, perhaps similar to that of swollen CNV. We show that an engineered CNV mutant (Pro73Gly) in which a conserved proline residue (Pro73) in the β-annulus of the CP arm is changed to glycine is resistant to proteolysis following in vitro zoospore binding assays. Moreover, Pro73Gly particles are transmitted only poorly by O. bornovanus . Together, the results of these studies suggest that CNV undergoes conformational change upon zoospore binding and that the conformational change is important for CNV transmissibility.

Propagation of yeast prions.

Prion proteins have been implicated in various human neurodegenerative disorders and form amyloid deposits in the diseased brain. Uniquely, prion proteins seem to be able to propagate this altered conformational state, generating more of the prion form of the protein and acting as infectious agents. The discovery in yeast of prion proteins that can be inherited stably through generations of cell division provides us with an experimental model that is allowing the mysteries of how prions are propagated to be unravelled.

Pulsed-alkylation mass spectrometry for the study of protein folding and dynamics: development and application to the study of a folding/unfolding intermediate of bacterial luciferase.

A new method employing the classical techniques of chemical modification of proteins and the new technology of mass spectrometry, known as pulsed-alkylation mass spectrometry (PA/MS), has been developed to probe the dynamic structure of folding intermediates and folded complexes of proteins under a variety of conditions. This method is fast and simple, and the results are easily interpreted. PA/MS may provide an alternative to H/D exchange monitored either by NMR or by electrospray ionization mass spectrometry for some experiments; for others, it may provide access to questions not readily answered by available methods. The objective of PA/MS is to determine simultaneously the location and the extent of labeling of functional groups in a protein by measuring the reactivity of cysteines with N-ethylmaleimide, within the context of the conformation of the protein under specific conditions. The method can also be applied to chemical modification of other amino acid residues employing any of a vast array of reagents, depending upon the specifics of the protein under investigation. The enormous range of reactivity of the thiol groups of the cysteinyl residues in proteins and the change in reactivity upon denaturation or conformational rearrangement afford a large signal change that can be correlated with changes in accessibility of the thiol group. The information obtained from the correlation of observed thiol reactivity with the local environment of each cysteinyl residue in the structure of the folded protein can be supplemented by results obtained from fluorescence, circular dichroism, or other methods, to develop an understanding of the structure and dynamics of altered conformational states. With bacterial luciferase as a model system, we have applied PA/MS to investigate the structural differences between the native heterodimeric enzyme and a folding intermediate that is well-populated in 2 M urea. The thiol residues at positions 307, 324, and 325 of the alpha subunit were much more reactive with N-ethylmaleimide in the presence of 2 M urea than in the native enzyme, suggesting that the C-terminal region of the alpha subunit was less tightly packed in the folding intermediate. The apparent unfolding of the C-terminal region of the alpha subunit of the alphabeta structure in 2 M urea appears to mimic the unfolding of the C-terminal domain of the free alpha subunit, also in 2 M urea, described by Noland, B. W., Dangott, L. J., and Baldwin, T. O. (1999) Biochemistry 38, 16136-16145. The approach described here should be applicable to a wide array of problems that have in common the need to determine the locations of conformational changes in proteins. Application of PA/MS to the investigation of the relative thermodynamic stability of the coordination complexes of zinc within each of the six zinc-finger domains of MRE-binding transcription factor-1 (Zn(6) MTF-zf) in its free and DNA-bound forms is presented in the companion paper in this issue [Apuy, J. L., Chen, X., Russell, D. H., Baldwin, T. O., and Giedroc, D. P. (2001) Biochemistry 40, 15164-15175].

Mutations in the Hinge of a Dynamic Loop Broadly Influence Functional Properties of Fructose-1,6-bisphosphatase

Loop 52-72 of porcine fructose-1,6-bisphosphatase may play a central role in the mechanism of catalysis and allosteric inhibition by AMP. The loop pivots between different conformational states about a hinge located at residues 50 and 51. The insertion of proline separately at positions 50 and 51 reduces k(cat) by up to 3-fold, with no effect on the K(m) for fructose 1,6-bisphosphate. The K(a) for Mg(2+) in the Lys(50) --> Pro mutant increases approximately 15-fold, whereas that for the Ala(51) --> Pro mutant is unchanged. Although these mutants retain wild-type binding affinity for AMP and the fluorescent AMP analog 2'(3')-O-(trinitrophenyl)adenosine 5'-monophosphate, the K(i) for AMP increases 8000- and 280-fold in the position 50 and 51 mutants, respectively. In fact, the mutation Lys(50) --> Pro changes the mechanism of AMP inhibition with respect to Mg(2+) from competitive to noncompetitive and abolishes K(+) activation. The K(i) for fructose 2,6-bisphosphate increases approximately 20- and 30-fold in the Lys(50) --> Pro and Ala(51) --> Pro mutants, respectively. Fluorescence from a tryptophan introduced by the mutation of Tyr(57) suggests an altered conformational state for Loop 52-72 due to the proline at position 50. Evidently, the Pro(50) mutant binds AMP with high affinity at the allosteric site, but the mechanism of allosteric regulation of catalysis has been disabled.

Prions are copper-binding proteins

Prions have become notorious in the past few years as potentially infectious particles implicated in a variety of spongiform encephalopathies, including Creutzfeld–Jacob syndrome, scrapie and `mad cow disease'[1], and certain serious psychiatric disorders[2]. Remarkably, these prion diseases are thought to be due to the prion protein (PrPC) achieving an altered conformational state (PrPSc), relative to the normal structure[1]. Little is known, however, about the function of prions in their normal (PrPC) form. Indeed mice lacking the gene that encodes the prion protein (Prnp−/− mice) develop quite normally, at least for the first 70 weeks of their (typically) 100-week life span[3, 4]. Such mice do show subtle aberrations, such as impaired γ-aminobutyric acid type A (GABAA)-receptor-mediated fast inhibition and long-term potentiation in the hippocampus[5], and altered circadian rhythms[6], but they appear clinically normal and are able to reproduce. As they age, however, mice of certain prion-deficient strains show progressive signs of ataxia that lead to premature death[4]—although Weissmann[7]has pointed out that this might be a consequence of additional deletions in these strains. In any case, there is speculation that the prion protein is somehow involved in senile dementias[1], with or without additional cofactors[8].